Journal of Physical Activity and Health, 2015, 12, 579 -587 http://dx.doi.org/10.1123/jpah.2013-0059 © 2015 Human Kinetics, Inc.
ORIGINAL RESEARCH
Effects of Yogic Practice on Metabolism and Antioxidant–Redox Status of Physically Active Males Rameswar Pal, Som Nath Singh, Kaushik Halder, Omveer Singh Tomer, Awadh Bihari Mishra, and Mantu Saha Background: This study was conducted to evaluate the effects of yogic practice on resting metabolism and redox status. Methods: The study was conducted on 64 physically trained male volunteers selected randomly at the Air Force Academy. The yoga group (n = 34) practiced yogasana, pranayama, and meditation for 3 months (February–May 2011) and the control group (n = 30) performed physical training. Antioxidant variables in blood samples along with physiological parameters were estimated before and after 3 months. Results: No significant difference was noted between baseline data of the control group and yoga group. Reduced glutathione, vitamin C, and vitamin E; the ratio of reduced to oxidized glutathione; and total antioxidant status were increased significantly following yogic practice. Activities of superoxide dismutase, glutathione S-transferase, and glutathione reductase were significantly increased, whereas activity of glutathione peroxidase was significantly decreased following yogic practice. Oxidized glutathione decreased significantly following yogic practice. A nonsignificant decrease of hydroperoxides, protein carbonyl, malondialdehyde, and blood sugar was noted in the yoga group. Carbon dioxide elimination and peripheral oxygen saturation increased significantly following yogic practice. No significant changes were observed in the control group following 3 months of physical training. Conclusions: Regular yogic practice can improve resting metabolism and redox status of the practitioner. Keywords: yoga, oxidative stress, oxygen consumption The word yoga derived from Sanskrit root yuj, meaning “union.” Yoga helps to build up better psychophysiological health, emotional harmony, and pranic balance by the reducing mental stress.1,2 Practice of asanas, pranayamas, and meditation provides the practitioner’s psychophysiological fitness and holistic health.3–5 Several studies have reported improvement in physiological, physical, and psychological functions following yogic practice.3–6 It has been reported that yoga improves cardio respiratory functions, aerobic capacity, and body composition by decreasing fat and increasing lean body mass, which also has a profound effect anaerobic power and anaerobic threshold.7–10 Yoga has a great beneficial effect on the autonomic nervous system by altering the low-frequency and high-frequency components of heart rate variability and improving gamma amino butyric acid.11,12 Earlier studies demonstrated that yogic practice has a beneficial role to decrease epinephrine and norepinephrine levels, which improves the autonomic function as the practioner goes toward parasympathodominance.13,14 Yoga also reduced stress and inflammation.15,16 Studies showed that yoga can decrease oxidative stress and improve antioxidant and redox status.17 Study showed 6-month yogic practice improved glutathione, as well as improved total antioxidant status.18 Studies have reported that 10 days of the Yoga-based Lifestyle Modification Program (YLMP) can decrease the serum Pal and Mishra are with the Centre for Advanced Research and Training in Yoga (CARTY), Defence Institute of Physiology and Allied Sciences (DIPAS), Timarpur, Delhi, India. Singh is with the Nutrition Division, Defence Institute of Physiology and Allied Sciences (DIPAS), Timarpur, Delhi, India. Halder, Tomer, and Saha ([email protected]) are with the Work Physiology and Yoga Division, Defence Institute of Physiology and Allied Sciences (DIPAS), Timarpur, Delhi, India.
concentration of thiobarbituric-acid-reactive substances, a marker of oxidative stress.19 The yogic practice for 10 weeks has a profound effect on plasma lipid peroxidation level and superoxide dismutase (SOD) activity.20 Hatha yoga exercises help in Type 2 diabetes mellitus by decreasing fasting blood glucose and improve lipid profile markers.21–23 Several experimental animal studies show that endurance exercise has a profound effect on oxidative stress. Endurance exercises reduce oxidative stress, activity of antioxidant, and defense enzymes in rats.24–28 Studies show that trained subjects have an increased activity of antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase as a result of exercise training.29 But studies regarding the effect of selected yogic practice on antioxidant and redox status along with physiological variables such as resting oxygen consumption (VO2) and resting energy expenditure (REE) on physically active individuals are rare. Hence, the current study was carried out to evaluate the effect of practicing yoga asanas, pranayama, and meditation on redox homeostasis, VO2, and REE on physically active males.
Materials and Methods Study Volunteers A total of 70 healthy and physically trained males from Indian Air Force personnel volunteered for the study. They were randomly divided into 2 groups: yoga (n = 34) and control (n = 30) groups. Initially, there were 35 volunteers in each group, but 1 volunteer from the yoga group and 5 volunteers from the control group dropped from the study and could not continue the entire schedule. Thus, the control group and yoga group completed the study with 30 and 34 participants, respectively. Participants were briefed about study protocol approved by the Institutional Ethics Committee, and 579
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individual written consent was obtained. They had a similar pattern of daily activities and were taking food from common mess. Along with their routine activities, the yoga group practiced yogasana, pranayama, and meditation for 3 months in the morning for duration of 1 hour. The control group did not change its lifestyle. As the participants were Indian Air Force personnel, they were physically trained and had undergone regular physical training as per their daily schedule. The yoga group practiced yoga with physical training. The control group had undergone only the physical training. The randomization and the schedule of parameters recording has been illustrated in Figure 1.Neither of the groups had any previous exposure to yogic exercises/practices. The anthropometric and physiological parameters of the yoga group and control group were recorded in the field laboratory between 7 AM and 9 AM in the months of February and May 2011. Blood samples were collected before and after the 3-month yoga training period of the yoga practice; blood samples from the control group were also collected at the same time for biochemical estimation.
Yoga Protocol The participants were practiced in selected yogic asanas, pranayama, and meditation for 6 days in a week. Yoga training was imparted by a qualified yoga instructor. The training consisted of 1 practical session (60 minutes). The yogic schedule is depicted in Table 1 and Figure 2.
Anthropometric Measurements Anthropometric measurements of the subjects were taken with minimal clothing (only inner wears). Body weight in kilograms was measured using an electronic weighing machine (Delmar, India) with least count of 0.1 kg. The standing (vertical) body height in
centimeters was measured by means of an anthropometer from the sole of the feet to the vertex while standing erect. Body Mass Index (BMI) was calculated as the ratio of weight in kilograms to a height in meters squared (kg/m2).
Resting Physiological Parameters VO2 and REE were measured by portable computerized equipment (K4b2 COSMED, Italy). The standard gas mixture was used to calibrate the oxygen and carbon dioxide. Respiratory quotient (RQ) and resting CO2 elimination were also obtained from the K4b2 system. Peripheral oxygen saturation (SpO2) was measured by using pulse oximetry with a probe attached to the index finger.
Biochemical Estimation Collection of Samples. Fasting venous blood samples were collected in ethylenediamine-tetraacetic-acid-treated vials from an antecubital vein in the morning when the volunteers were in resting condition in the lying position. Just after collection, 1 portion of ethylenediaminetetraacetic acid–treated whole blood was kept in a separate vial. The aliquot of 0.5 ml of whole blood was treated with equal volume of 10% metaphosphoric acid and 1 ml 10% trichloroacetic acid (TCA) was mixed properly and kept in a separate vial to analyze vitamin C, reduced glutathione (GSH), oxidized glutathione (GSSG), and malondialdehyde (MDA). The remaining blood sample was centrifuged at 1000 g for 15 minutes to collect plasma and red blood cell (RBC) pellets. RBCs were washed twice with a cold 150 mM potassium chloride solution at 4°C. All the chemicals for biochemical estimation have been purchased from Sigma Aldrich Co. GSH and GSSG. Supernatant obtained from metaphosphoric acid
and TCA-treated whole blood was used to estimate the GSH and GSSG by using a fluorimetric method.30 A total of 10 µl supernatant
Figure 1 — Schematic representation of study design. JPAH Vol. 12, No. 4, 2015
Yoga on Metabolism and Antioxidant–Redox Status 581
Table 1 Details of Yogic Package Was Practiced by the Volunteers Cleansing processes: session = 2 min 1. Kapalbhati (Rapid shallow breathing): It is a vigorous hyperventilation practice, which involves forceful exhalation and passive inhalation.
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Yogasanas (yogic postures): session = 40 min 1. Suryanamaskar: Sun salutation in 12 different postures, 1 round 2. Padmasana: In Sanskrit, “Padma” means “lotus.” This asana in its final position resembles the petals of a lotus. 3. Yogamudra 4. Matsayasana: In Sanskrit, “matsya” mean “fish.” That is why it is known as Matsyasana (fish posture) 5. Suptapavanmuktasana: The Sanskrit word “pawan” means “wind” and the word “mukta” means “release” or “free.” Hence, this is a wind-releasing pose because it is very useful in removing wind or flatulence from the intestines and stomach. 6. Pavanmuktasana: “Pawana” means “wind” and “mukta” means to “release.” When it is practiced, it helps to release excessive gases from the body. 7. Paschimottanasana: “Paschim” means “back” and “uttan” means “stretching.” 8. Vajrasana: Sitting posture 9. Suptavajrasana 10. Gomukhasana: The interlocked hand in this asana takes the shape of Gomukh, which means a cow’s face in Sanskrit. That is why it is known as Gomukhasana. 11. Sarvangasana: In Sanskrit, “Sarvang” means “all parts of the body.” Because this asana having the effect on almost all the body parts and organs, it is called Sarvangasana. 12. Halasana: “Hala” means a “plow” in Sanskrit. The pose is called Halasana because in its practice, the body takes the shape of Indian plow. 13. Karnapedasana 14. Bhujangasana: In Sanskrit, “Bhujanga” means “cobra.” The final position of this asana resembles the “hooded snake.” Hence, it is called Bhujangasana. 15. Shavasana: In Sanskrit, “shava” means a “dead body.” Relaxed supine posture. This asana resembles a dead body that is why it is known as Shavasana. Pranayama (breathing exercises): session = 10 min 1. Bhastrika: Forceful expulsion of breathing, Bhastrika pranayama is all about inhaling and exhaling completely so that your body gets maximum amount of oxygen. 2. Anulom—vilom: Anuloma viloma is also called the alternate nostril breathing. 3. Bhramri: The word “Bhramari” is derived from “Bhramara,” which means a black bee. While practicing this Pranayama, the sound produced resembles the buzzing of a black bee with closed eyes, ears, and lips. Meditation: session = 8 min 1. Omkar meditation: Om chant
was treated with 100 µl o-phthalaldehyde at pH 8 and estimated GSH using a Fluorescence spectrophotometer with an excitation and emission wavelength of 350 nm and 420 nm, respectively. GSSG was also measured with this same procedure at pH 12 after treatment with 10 µl N-ethylmaleimide to prevent interference of GSH with the measurement of GSSG. Total Antioxidant Status (TAS). It was measured in 10-µl plasma with an additional 1.0 ml of ABTS (2,2'-Azino-di-[3ethylbenzthiazoline sulphonate]) radical cation decolorizing diluted reagent, using a Randox kit (Cat No NX 2332; Randox Laboratories, UK) Vitamin C. Supernatant obtained from metaphosphoric acid and TCA-treated whole was used to estimate vitamin C.31 Briefly, the 0.5 ml supernatant was treated with a few drops (0.050 ml) of bromine water followed by a few drops (50 µl) of thiourea to remove the excess bromine, and thus, the clear solution was obtained. Then 0.4 ml 2,4-dinitrophenyl-hydrazine solution was added to all standards and also sampled with the oxidized ascorbic acid. Total vitamin C content employing coupling reaction of 2,4-dinitrophenyl hydrazine (DNPH) was measured at 540 nm using a spectrophotometer.
Vitamin E. The estimation of vitamin E from 100 µl of plasma
using the bathophenanthroline method.32 The lipid residue obtained using 100 µl of redistilled ethanol and 500 µl of petroleum ether was redissolved in 400 µl of absolute ethanol. To this solution, 50 µl ferric chloride, 50 µl orthophosphoric acid, and 50 µl bathophenanthroline reagents were added. Vitamin E presents in the lipid residue, reduces ferric ions to ferrous ions, and forms a pink-colored complex with bathophenanthroline orthophosphoric acid. Absorption due to the pink complex was measured at 536 nm using a spectrophotometer.
Hydroperoxide Estimation (FOX 1 Assay). Hydroperoxide was estimated from plasma.33 Xylenol orange, in an acidic solution with sorbitol, and ammonium iron sulfate reacted to produce a purple color proportional to the concentration of hydrogen peroxide (H2O2) in the sample. We took 50µl of the sample plasma and added 950 µl of FOX reagent and incubate for 30 min at room temperature. We read the absorbance at a web length of 560 nm. Protein Carbonyl (PC). PC reacts with DNPH to form a Schiff
base to produce the corresponding hydrazine, which can be analyzed spectrophotometrically.34 A plasma sample (50µl) was added to 0.950 ml of DNPH and mixed thoroughly. After proper mixing,
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Figure 2 — Various yoga postures as practiced by the volunteers during yoga session.
1.0 ml of TCA was added. After 3 times washing of the precipitate obtained from above mixture, 2 ml of guanidine hydrochloride was added and optical density (OD) was measured at 366 nm web length using a spectrophotometer. Malondialdehyde (MDA). MDA content of the sample was estimated by using plasma.35 In brief, metaphosphoric acid and TCA-treated whole blood supernatant were mixed with equal volumes of thiobarbituric acid solution and incubated at 90°C for 30 minutes; color density was measured spectrophotometrically at 535 nm. Glutathione Reductase (GR). RBC lysate was used to measure
the GR activity.36 The final reaction mixture contains GSSG and NADPH; these are incubated at pH 7.5, and the change in OD was measured at 340 nm for 3 minutes at an interval of 30 seconds.
Glutathione Peroxidase (GPx). GPx was measured in RBC
pellets by using the Cayman Kit (Cat No. 703102, Cayman Chemical Laboratories, USA). Assay buffer (100 µl) and cosubstrate was added to all the wells of enzyme-linked immunosorbent assay plates. A total of 20 µl of diluted GPx control was added to the positive control wells, and 20 µl of the sample was added to the sample wells. An enzymatic reaction was initiated by adding 20 µl of cumene hydroxide. Change in OD was recorded at 60-second intervals for 300 seconds at 340 nm using an enzyme-linked immunosorbent assay reader. Calculation was made according the kit protocol.
Superoxide Dismutase (SOD). SOD was measured in RBC
pellets by using the Cayman Kit (Cat No. 706002, Cayman Chemical Laboratories). A total of 200 µl of diluted radical was added to all
wells of the plate. Ten µl of standard and 10 µl of sample added, respectively, to the standard wells and sample wells. An enzymatic reaction was initiated by adding 20 µl of xanthine oxidase. OD was measured after a 20-minute incubation at 460 nm. Calculation was made according to kit protocol. Catalase. Catalase was measured spectrophotometrically using
RBC lysate.37 Briefly, a 50 µl lysate of RBC was treated with 1.0 ml of 30-mM H2O2, and absorbance was measured at a 240-nm wavelength for 1 minute at an interval of 30 seconds.
G l u t a t h i o n e S Tr a n s fe r a s e ( G S T ) . T h e e s t i m a t i o n
of GST using 1-chloro-2,4-dinitrobenzene was carried out spectrophotometrically.38 The reaction mixture contains 0.1 ml of GSH, 20 µl of 1-chloro-2, 4-dinitrobenzene and 1 ml of a phosphate buffer. The reaction was initiated by adding 0.1 ml of RBC lysate. The change in OD at an interval of 15 seconds for 300 seconds was recorded using a spectrophotometer at 340 nm. Glucose-6-Phosphate Dehydogenase (G6PDH). The estimation
of G6PDH was carried out using RBC lysate.39 A total of 100 µl of RBC lysate was incubated with 100 mM of magnesium chloride (200 µl), glucose-6-phosphates (100 µl), nicotinamide adenine dinucleotide phosphate (100 µl), and a buffer at pH 7.4. The change in OD was measured at 340 nm for 3 minutes at an interval of 30 seconds using a spectrophotometer.
Blood Glucose. Blood glucose was estimated by using GlucoDr
(All Medicus Co., Ltd., Korea), blood glucose test meter in fasting condition between 6:30 AM and 7:15 AM, and the middle finger tip was used to take the blood.
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Statistical Analysis One-way analysis of variance (ANOVA) without replication to analyze significance of between-group variance and within-group therein was performed. Once Schiff’s F showed P ≤ .05, data underwent the Tukey–Kramer post hoc test for within-group significance, using GraphPad InStat Version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). Data was expressed as mean ± standard error of the mean (SEM). P ≤ .05 was considered to be statically significant.
Results
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Anthropometric Data Anthropometric data are summarized in Table 2. Body weight (kg) and BMI (kg/m2) did not show any significant change before and after yogic practices, though a nonsignificant reduction in both these parameters was observed in the yoga group, whereas there was an increase in these parameters the control group following the period of yogic practice. No significant difference was found between the baseline data of the control group and yoga group.
Resting Physiological Variables Resting physiological variables are illustrated in Table 3. VO2 and REE were decreased in the yoga group following 3 months of yogic practices. On the other hand, RQ (P < .01) and resting CO2 elimination (P < .001) decreased significantly as a result of yogic practices. SpO2 was also significantly increased following 3 months of yogic practice. No significant change was noted in the control group following yogic practice. No significant difference was noted between baseline data of the control group and yoga group in these resting physiological parameters.
Enzymatic and Nonenzymatic Antioxidant and Blood Glucose Nonenzymatic antioxidant variables and blood sugar are depicted in Table 4. The levels of GSH were increased significantly (P < .001), with a decrease in GSSG (P < .01). This led to a significant (P < .001) increase in the GSH and GSSG ratio following 3 months of yogic practices. TAS, an important antioxidant marker, increased significantly (P < .001) following 3 months of yogic practice. The levels of vitamin C and vitamin E were also increased significantly (P < .001) following yogic practices. A nonsignificant decrease in MDA levels of the yoga group was noted. No significant changes were observed in levels of hydroperoxides measured using FOX , PC, and blood glucose levels following yogic practice, although a nonsignificant reduction in these parameters was observed. The control group did not show any significant change in nonenzymatic antioxidant variables following yogic practice. No significant difference was found between baseline data of the control group and yoga group in enzymatic and nonenzymatic antioxidant parameters and levels of blood glucose. Activities of antioxidant enzymes are summarized in Figure 3. SOD and GST activity increased significantly (P < .001), while GPx activity decreased significantly (P < .001). GR activity also increased significantly (P < .05) following yogic practice. No significant change was noted in these parameters in the control group. The catalase and G6PDH activity in the yoga group increased nonsignificantly as a result of yogic practice.
Discussion Body weight and BMI did not show any significant change in the current study, though there was a nonsignificant reduction in the mean values of both parameters in the yoga group. There are
Table 2 Physical Characteristics of Subjects Control group (n = 30) Age (years) Height (cm) Body weight (kg) Body mass index (kg/m2)
Yoga group (n = 34)
Before
After
Before
After
37.5 ± 1.39 171.7 ± 2.95 81.1 ± 6.79 27.4 ± 1.43
— — 81.4 ± 7.32 27.5 ± 1.68
40.9 ± 0.90 169.5 ± 0.90 73.8 ± 8.02 25.7 ± 0.43
— — 73.1 ± 8.20 25.4 ± 0.44
Note. Values are expressed as mean ± SEM.
Table 3 Physiological Changes of Control and Yoga Group Before and After 3 Months Control group (n = 30) Resting O2 consumption (ml/min) Resting CO2 elimination (ml/min) Respiratory quotient Resting energy expenditure (kj/min) Peripheral oxygen saturation (%)
Yoga group (n = 34)
Before
After
Before
After
238.5 ± 13.7 220.8 ± 11.6 0.92 ± 0.04 4.79 ± 0.24 97.3 ± 0.26
276.9 ± 17.68 229.6 ± 18.95 0.88 ± 0.03 5.53 ± 0.20 97.3 ± 0.42
240.7 ± 12.9 227.5 ± 13.1 0.95 ± 0.02 4.97 ± 0.27 96.3 ± 0.41
210.4 ± 8.8 173. 1 ± 8.2** 0.82 ± 0.02*** 4.20 ± 0.18 97.6 ± 0.15**
Note. Values are expressed as mean ± SEM. ** P < .01, as compared with yoga group base line data; *** P < .001, as compared with yoga group base line data. No significant difference was noted between base line data of control group and yoga group. JPAH Vol. 12, No. 4, 2015
Table 4 Antioxidant, Oxidative Stress Markers, and Blood Glucose of Control and Yoga Group Before and After 3 Months Control group (n = 30) GSH (nmol/ml) GSSG (nmol/ml) GSH/GSSG TAS (mmol/ml) Vitamin C (mg/dl)
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Vitamin E (μg/ml) Hydroperoxides (μmol/ml) MDA (nmol/ml) PC (nmol/ml) Blood glucose (mg/dl)
Yoga group (n = 34)
Before
After
Before
After
260.2 ± 17.3 399.1 ± 10.13 0.65 ± 0.06 2.66 ± 0.15 1.37 ± 0.08 12.24 ± 1.97 506.1 ± 37.1 8.56 ± 0.63 118.6 ± 12.71 101.3 ± 5.3
264.7 ± 15.6 392.7 ± 14.1 0.67 ± 0.07 2.78 ± 0.09 1.34 ± 0.06 11.80 ± 2.73 509.4 ± 36.9 8.38 ± 0.60 117.6 ± 13.3 101.7 ± 3.9
292.4 ± 18.5 394.9 ± 19.4 0.98 ± 0.25 2.69 ± 0.06 1.44 ± 0.08 13.02 ± 0.90 545.1 ± 50.6 7.78 ± 0.50 114.2 ± 17.8 96.4 ± 1.6
457.5 ± 16.7 *** 300.6 ± 18.3** 1.86 ± 0.21*** 3.19 ± 0.04*** 3.35 ± 0.12*** 19.62 ± 0.38*** 452.6 ± 60.2 6.01 ± 0.46 106.4 ± 9.7 95.7 ± 9.7
Note. Values are expressed as mean ± SEM. ** P < .01, as compared with yoga group base line data; *** P < .001, as compared with yoga group base line data. Abbreviations: GSH, reduced glutathione; GSSG, oxidized glutathione; GSH/GSSG, ratio of reduced and oxidized glutathione; TAS, total antioxidant status; MDA, malondialdehyde; PC, protein carbonyl.
Figure 3 — Changes in enzymatic activity of control and yoga group before and after 3 months. (A) Super oxide dismutase, (B) catalase, (C) glutathione peroxidase, (D) glutathione reductase, (E) glutathione 6 phosphate dehydrogenase, and (F) glutathione S-transferase. Values are expressed as mean ± SEM. ** P < .01, as compared with yoga group base line data; *** P < .001, as compared with yoga group base line data.
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conflicting reports in body weight changes after yogic exercises in the literature, as both a decrease and an increase are reported.8,40 A possible explanation could be that the participants of the current study were trained Air Force personnel and were habituated to perform routine physical exercises. During the study, the yoga group performed yoga training in place of exercise. The VO2, resting CO2 elimination, RQ, and REE decreased as a result of yogic practice. Various studies showed that relaxation techniques decreased O2 consumption, CO2 elimination, and respiration rate.41,42 Yoga may have a beneficial role to calm down the body and mind, which decreases O2 consumption as well as CO2 elimination along with decreasing REE as a relaxation response. The SpO2 of the body increased due to yogic practices, and there was no such change observed in the control group. This may due to the breathing maneuvers such as Kapalbhati, Bhastrika, Anulom-vilom, and Bhramari.43,44 This study shows that SOD activity increases significantly, and there is a trend to increase in catalase activity in the yoga group, whereas SOD activity decreases in the control group. Earlier studies have reported that the yogic practice helped in the increase of SOD activity.21 Yogic practices decreased the activity of GPx and increased activity of GR. The GSH level was increased significantly in the yoga group; the ratio of reduced to oxidized glutathione (GSH/ GSSG ratio) is another important and sensitive marker of the antioxidant system that has been shown to increase significantly in the yoga group. These may be due to improvement of the antioxidant defense mechanism by yoga.18 H2O2 is generated directly from superoxide (O2–) though a rapid dismutation reaction that can occur either enzymatically with SOD or spontaneously. This means that wherever O2– is generated, there is also the formation of H2O2. SOD and catalase are metalloproteins that catalyze O2– and H2O2, respectively. SOD catalyzes the formation of H2O2 from 2 O2–, whereas catalase catalyzes the formation of oxygen and water from an H2O2 molecule. Increments of SOD activity produced more H2O2. Catalase activity was increased, perhaps because yogic practice catalyzed more H2O2 and produced O2 and water. Hence, hydroperoxide levels were decreased in the yoga group. GPx catalyzes the reduction of hydroperoxides, including H2O2, and functions to protect the cell from oxidative damage. GSSG, produced upon reduction of hydroperoxide by GPx, is recycled to its reduced state by GR using NADPH as a hydrogen donor. Here, the production of NADPH is coupled with G6PDH. This study showed that yoga increased GSH and decreased GSSG by altering the activity of GPx, GR, and G6PDH. An earlier report also supports this observation.18 GSTs are multifunctional enzymes, which play a key role in cellular detoxification. The enzymes protect cells against toxicants by conjugating them with glutathione, thereby neutralizing their electrophilic sites and rendering the products more water-soluble.45 The glutathione conjugates are metabolized further to mercapturic acid and then excreted. In this study, GST activity increased significantly after 3 months of yogic practices. This also acts as a defense mechanism of the body against reactive oxygen species production. GST also acts to catalyze the reaction between the thiol group of GSH and possible alkylating agents, allowing GSH to carry out its detoxifying function. Levels of vitamins C and E increased in the yoga group. Vitamin C and vitamin E have a profound effect on the body; during lipid peroxidation, vitamins function as a potent chain-breaking antioxi-
dant, intercepting lipid peroxyl radicals and forming the vitamin E radical as a product. Vitamin C acts to generate vitamin E by accepting the electron from the vitamin E radical, with the vitamin C radical being formed, and either in the urine or regenerated to vitamin C via electron donation from GSH. It also reported that both vitamin E and C function to scavenge superoxide radical and hydroxyl radical in lipid and aqueous phase, respectively, inhibiting lipid peroxidation and oxidative damage to other macromolecules. Without changing any diet pattern, vitamin C and vitamin E were increasing significantly in the yoga group. This may be due to an increase in total antioxidant status or shifting of the volunteers toward reducing redox status. TAS has been seen to improve significantly after yogic practice, denoting a marked improvement in the overall cellular antioxidant level. Studies reported that the yogic practice helps in the improvement of TAS.18 Another study also showed that diaphragmatic breathing/breathing maneuvers also increased biological antioxidant potential and reduced exercise-induced oxidative stress.17 Oxidative stress generally causes damage to the membrane polyunsaturated fatty acids leading to the generation of MDA, a thiobarbituric-acid-reacting substance. This biochemical study implies that the products of lipid peroxidation decrease with yogic practice. This is also supported by several previous studies.20–22 Studies also showed that the yogic practice decreased MDA level in diabetic patients.21 Sudarshan Kriya yoga has been reported to be beneficial in decreasing MDA level.46 Protein carbonyl also decreased due to yogic practice indicating prevention of protein oxidation. Blood sugar levels decreased in the yoga group after 3 months of yogic practice, whereas no changes were observed in the blood sugar levels of the control group. There are reports suggesting that blood sugar levels decreased in the diabetic patient with yogic practices.22
Conclusion This study showed that yoga has the beneficial role to decrease oxidative stress by improving enzymatic defense mechanism and nonenzymatic antioxidants. It also maintains antioxidant and redox status. Yoga has also a beneficial role in relaxing the body by decreasing VO2 and REE. Acknowledgments Sincere gratitude to the director of the Defence Institute of Physiology and Allied Sciences (DIPAS) in Delhi, India, for giving necessary permission to conduct this study. We are thankful to Director Morarji Desai, National Institute of Yoga (MDNIY), New Delhi. We are also grateful to volunteers of this study for their kind help in the successful completion of the study. We acknowledge the support of staff members of the CARTY and nutrition division of DIPAS. The study was financially supported by DIPAS and MDNIY.
References 1. Pal R, Saha M. Role of yogic practice on physical health: a review. Indian J Appl Research. 2013;3(4):34–36. 2. Field T. Yoga clinical review. Complement Ther Clin Pract. 2011;17(1):1–8. PubMed 3. Sengupta P. Health impacts of yoga and pranayama: a state-of-the-art review. Int J Prev Med. 2012;3(7):444–458. PubMed
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586 Pal et al 4. Chan W, Immink MA, Hillier S. Yoga and exercise for symptoms of depression and anxiety in people with poststroke disability: a randomized, controlled pilot trial. Altern Ther Health Med. 2012;18(3):34–43. PubMed 5. Chung SC, Brooks MM, Rai M, Balk JL, Rai S. Effect of sahaja yoga meditation on quality of life, anxiety, and blood pressure control. J Altern Complement Med. 2012;18(6):589–596. PubMed doi:10.1089/ acm.2011.0038 6. Gupta SS, Sawane MV. A comparative study of the effects of yoga and swimming on pulmonary functions in sedentary subjects. Int J Yoga. 2012;5(2):128–133. PubMed doi:10.4103/0973-6131.98232 7. Balasubramanian B, Pansare MS. Effect of yoga on aerobic and anaerobic power of muscles. Indian J Physiol Pharmacol. 1991;35(4):281– 282. PubMed 8. Bera TK, Rajapurkar MV. Body composition, cardiovascular endurance and anaerobic power of yogic practitioner. Indian J Physiol Pharmacol. 1993;37(3):225–228. PubMed 9. Ray US, Sinha B, Tomer OS, Pathak A, Dasgupta T, Selvamurthy W. Aerobic capacity & perceived exertion after practice of Hatha yogic exercises. Indian J Med Res. 2001;114:215–221. PubMed 10. Ray US, Mukhopadhyaya S, Purkayastha SS, et al. Effect of yogic exercises on physical and mental health of young fellowship course trainees. Indian J Physiol Pharmacol. 2001;45(1):37–53. PubMed 11. Streeter CC, Gerbarg PL, Saper RB, Ciraulo DA, Brown RP. Effects of yoga on the autonomic nervous system, gamma-aminobutyric-acid, and allostasis in epilepsy, depression, and post-traumatic stress disorder. Med Hypotheses. 2012;78(5):571–579. PubMed doi:10.1016/j. mehy.2012.01.021 12. Muralikrishnan K, Balakrishnan B, Balasubramanian K, Visnegarawla F. Measurement of the effect of Isha Yoga on cardiac autonomic nervous system using short-term heart rate variability. J Ayurveda Integr Med. 2012;3(2):91–96. PubMed doi:10.4103/0975-9476.96528 13. Selvamurthy W, Sridharan K, Ray US, et al. A new physiological approach to control essential hypertension. Indian J Physiol Pharmacol. 1998;42(2):205–213. PubMed 14. Murugesan R, Govindarajulu N, Bera TK. Effect of selected yogic practices on the management of hypertension. Indian J Physiol Pharmacol. 2000;44(2):207–210. PubMed 15. Yadav RK, Magan D, Mehta N, Sharma R, Mahapatra SC. Efficacy of a short-term yoga-based lifestyle intervention in reducing stress and inflammation: preliminary results. J Altern Complement Med. 2012;18(7):662–667. PubMed doi:10.1089/acm.2011.0265 16. Kiecolt-Glaser JK, Christian L, Preston H, et al. Stress, inflammation, and yoga practice. Psychosom Med. 2010;72(2):113–121. PubMed doi:10.1097/PSY.0b013e3181cb9377 17. Martarelli D, Cocchin M, Scuri S, Pompei P. Diaphragmatic breathing reduces exercise-induced oxidative stress. Evid Based Complement Alternat Med. 2011;932430. doi:10.1093/ecam/nep169 18. Sinha S, Singh SN, Monga YP. Ray US. Improvement of glutathione and total antioxidant status with yoga. The J Alt Com Med. 2007;13(10):1085–1090. doi:10.1089/acm.2007.0567 19. Yadav RK, Ray RB, Vempati R, Bijlani R. Effect of a comprehensive yoga based lifestyle modification programme on lipid peroxidation. Indian J Physiol Pharmacol. 2005;49(3):358–362. PubMed 20. Bhattacharya S, Pandey US, Verma NS. Improvement in oxidative status with yogic breathing in young healthy males. Indian J Physiol Pharmacol. 2002;46:349–354. PubMed 21. Gordon LA, Morrison EY, McGrowder DA, et al. Effect of exercise therapy on lipid profile and oxidative stress indicators in patients with type 2 diabetes. BMC Complement Altern Med. 2008;8:21. PubMed doi:10.1186/1472-6882-8-21
22. Malhotra V, Singh S, Sharma SB, et al. The status of NIDDM patients after yoga asanas: assessment of important parameters. J Clin and Diagno Res. 2010;4:2652–2667. 23. Singh S, Kyizom T, Singh KP, Tandon OP, Madhu SV. Influence of pranayamas and yoga: asanas on serum insulin, blood glucose and lipid profile in type 2 diabetes. Indian J Clin Biochem. 2008;23(4):365–368. PubMed doi:10.1007/s12291-008-0080-9 24. Venditti P, Masullo P, Di Meo S. Effect of training on H2O2 release by mitochondria from rat skeletal muscle. Arch Biochem Biophys. 1999;372(2):315–320. PubMed doi:10.1006/abbi.1999.1494 25. Leeuwenburgh C, Fiebig R, Chandwaney R, Ji LL. Aging and exercise training in skeletal muscle: responses of glutathione and antioxidant enzyme systems. Am J Physiol. 1994;267(2 Pt 2):439–445. PubMed 26. Leeuwenburgh C, Hollander J, Leichtweis S, Griffiths M, Gore M, Ji LL. Adaptations of glutathione antioxidant system to endurance training are tissue and muscle fiber specific. Am J Physiol. 1997;272(1 Pt 2):363–369. PubMed 27. Powers SK, Criswell D, Lawler J, et al. Influence of exercise and fiber type on antioxidant enzyme activity in rat skeletal muscle. Am J Physiol. 1994;266(2 Pt 2):375–380. PubMed 28. Powers SK, Criswell D, Lawler J, et al. Rigorous exercise training increases superoxide dismutase activity in ventricular myocardium. Am J Physiol. 1993;265(6 Pt 2):2094–2098. PubMed 29. Somani SM, Frank S, Rybak LP. Responses of antioxidant system to acute and trained exercise in rat heart subcellular fractions. Pharmacol Biochem Behav. 1995;51(4):627–634. PubMed doi:10.1016/0091-3057(94)00427-K 30. Hissin PJ, Hiff R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 1976;74(1):214– 226. PubMed doi:10.1016/0003-2697(76)90326-2 31. Khan MMR, Rahman MM, Islam MS, Begum SA. A simple ultraviolet—spectrometric method for the determination of vitamin C content in various fruits and vegetables at sylhet area in Bangladesh. J Biol Sci. 2006;6(2):388–392. doi:10.3923/jbs.2006.388.392 32. Desai ID. Vitamin E analysis method for animal tissues. Methods Enzymol. 1984;105:138–147. PubMed doi:10.1016/S00766879(84)05019-9 33. Wolff SP. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol. 1994;233:182–189. doi:10.1016/S0076-6879(94)33021-2 34. Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol. 1994;233:357– 363. PubMed doi:10.1016/S0076-6879(94)33041-7 35. Ohkawa H. Assay of lipid peroxide in animal tissue by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351–358. PubMed doi:10.1016/0003-2697(79)90738-3 36. Racker E. Glutathione reductase from baker’s yeast and beef liver. J Biol Chem. 1955;217(2):855–865. PubMed 37. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–126. PubMed doi:10.1016/S0076-6879(84)05016-3 38. Habig WH, Jakoby WB. Assay for differentiation of glutathione S-transferase. Methods Enzymol. 1981;77:398–405. PubMed doi:10.1016/ S0076-6879(81)77053-8 39. Bernardi L, Passino C, Spadacini G, et al. Reduced hypoxic ventilatory response with preserved blood oxygenation in yoga trainees and Himalayan Buddhist monks at altitude: evidence of a different adaptive strategy? Eur J Appl Physiol. 2007;99(5):511–518. PubMed doi:10.1007/s00421-006-0373-8 40. Kesterson J, Clinch NF. Metabolic rate, respiratory exchange ratio, and apneas during meditation. Am J Physiol. 1989;256(3 Pt 2):632–638. PubMed
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Yoga on Metabolism and Antioxidant–Redox Status 587 44. Mason H, Vandoni M, Barbieri G, Codrons E, Ugargol V, Bernardi L. Cardiovascular and respiratory effect of yogic slow breathing in the yoga beginner: what is the best approach? Evid Based Complement Alternat Med. 2013; 743504. doi:10.1155/2013/743504 45. Habig WH, Pabst MJ, Jakoby WB, Glutathione S. Transferases the first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249(22):7130–7139. PubMed 46. Agte VV, Tarwadi K. Sudarshan kriya yoga for treating type 2 diabetes: a preliminary study. Altern Complement Ther. 2004;10(4):220–222. doi:10.1089/1076280041580323
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41. Warrenburg S, Pagano RR, Woods M, Hlastala M. A comparison of somatic relaxation and EEG activity in classical progressive relaxation and transcendental meditation. J Behav Med. 1980;3(1):73–93. PubMed doi:10.1007/BF00844915 42. Boyland E, Chasseaud LF. The role of glutathione and glutathione S-transferases in mercapturic acid biosynthesis. Adv Enzymol. 1969;32:173–219. PubMed 43. Ashok C. Impact of asanas and pranayama on blood oxygen saturation level. Br J Sports Med. 2010;44:i69. doi:10.1136/ bjsm.2010.078725.228
JPAH Vol. 12, No. 4, 2015
ORIGINAL RESEARCH
Effects of Yogic Practice on Metabolism and Antioxidant–Redox Status of Physically Active Males Rameswar Pal, Som Nath Singh, Kaushik Halder, Omveer Singh Tomer, Awadh Bihari Mishra, and Mantu Saha Background: This study was conducted to evaluate the effects of yogic practice on resting metabolism and redox status. Methods: The study was conducted on 64 physically trained male volunteers selected randomly at the Air Force Academy. The yoga group (n = 34) practiced yogasana, pranayama, and meditation for 3 months (February–May 2011) and the control group (n = 30) performed physical training. Antioxidant variables in blood samples along with physiological parameters were estimated before and after 3 months. Results: No significant difference was noted between baseline data of the control group and yoga group. Reduced glutathione, vitamin C, and vitamin E; the ratio of reduced to oxidized glutathione; and total antioxidant status were increased significantly following yogic practice. Activities of superoxide dismutase, glutathione S-transferase, and glutathione reductase were significantly increased, whereas activity of glutathione peroxidase was significantly decreased following yogic practice. Oxidized glutathione decreased significantly following yogic practice. A nonsignificant decrease of hydroperoxides, protein carbonyl, malondialdehyde, and blood sugar was noted in the yoga group. Carbon dioxide elimination and peripheral oxygen saturation increased significantly following yogic practice. No significant changes were observed in the control group following 3 months of physical training. Conclusions: Regular yogic practice can improve resting metabolism and redox status of the practitioner. Keywords: yoga, oxidative stress, oxygen consumption The word yoga derived from Sanskrit root yuj, meaning “union.” Yoga helps to build up better psychophysiological health, emotional harmony, and pranic balance by the reducing mental stress.1,2 Practice of asanas, pranayamas, and meditation provides the practitioner’s psychophysiological fitness and holistic health.3–5 Several studies have reported improvement in physiological, physical, and psychological functions following yogic practice.3–6 It has been reported that yoga improves cardio respiratory functions, aerobic capacity, and body composition by decreasing fat and increasing lean body mass, which also has a profound effect anaerobic power and anaerobic threshold.7–10 Yoga has a great beneficial effect on the autonomic nervous system by altering the low-frequency and high-frequency components of heart rate variability and improving gamma amino butyric acid.11,12 Earlier studies demonstrated that yogic practice has a beneficial role to decrease epinephrine and norepinephrine levels, which improves the autonomic function as the practioner goes toward parasympathodominance.13,14 Yoga also reduced stress and inflammation.15,16 Studies showed that yoga can decrease oxidative stress and improve antioxidant and redox status.17 Study showed 6-month yogic practice improved glutathione, as well as improved total antioxidant status.18 Studies have reported that 10 days of the Yoga-based Lifestyle Modification Program (YLMP) can decrease the serum Pal and Mishra are with the Centre for Advanced Research and Training in Yoga (CARTY), Defence Institute of Physiology and Allied Sciences (DIPAS), Timarpur, Delhi, India. Singh is with the Nutrition Division, Defence Institute of Physiology and Allied Sciences (DIPAS), Timarpur, Delhi, India. Halder, Tomer, and Saha ([email protected]) are with the Work Physiology and Yoga Division, Defence Institute of Physiology and Allied Sciences (DIPAS), Timarpur, Delhi, India.
concentration of thiobarbituric-acid-reactive substances, a marker of oxidative stress.19 The yogic practice for 10 weeks has a profound effect on plasma lipid peroxidation level and superoxide dismutase (SOD) activity.20 Hatha yoga exercises help in Type 2 diabetes mellitus by decreasing fasting blood glucose and improve lipid profile markers.21–23 Several experimental animal studies show that endurance exercise has a profound effect on oxidative stress. Endurance exercises reduce oxidative stress, activity of antioxidant, and defense enzymes in rats.24–28 Studies show that trained subjects have an increased activity of antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase as a result of exercise training.29 But studies regarding the effect of selected yogic practice on antioxidant and redox status along with physiological variables such as resting oxygen consumption (VO2) and resting energy expenditure (REE) on physically active individuals are rare. Hence, the current study was carried out to evaluate the effect of practicing yoga asanas, pranayama, and meditation on redox homeostasis, VO2, and REE on physically active males.
Materials and Methods Study Volunteers A total of 70 healthy and physically trained males from Indian Air Force personnel volunteered for the study. They were randomly divided into 2 groups: yoga (n = 34) and control (n = 30) groups. Initially, there were 35 volunteers in each group, but 1 volunteer from the yoga group and 5 volunteers from the control group dropped from the study and could not continue the entire schedule. Thus, the control group and yoga group completed the study with 30 and 34 participants, respectively. Participants were briefed about study protocol approved by the Institutional Ethics Committee, and 579
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individual written consent was obtained. They had a similar pattern of daily activities and were taking food from common mess. Along with their routine activities, the yoga group practiced yogasana, pranayama, and meditation for 3 months in the morning for duration of 1 hour. The control group did not change its lifestyle. As the participants were Indian Air Force personnel, they were physically trained and had undergone regular physical training as per their daily schedule. The yoga group practiced yoga with physical training. The control group had undergone only the physical training. The randomization and the schedule of parameters recording has been illustrated in Figure 1.Neither of the groups had any previous exposure to yogic exercises/practices. The anthropometric and physiological parameters of the yoga group and control group were recorded in the field laboratory between 7 AM and 9 AM in the months of February and May 2011. Blood samples were collected before and after the 3-month yoga training period of the yoga practice; blood samples from the control group were also collected at the same time for biochemical estimation.
Yoga Protocol The participants were practiced in selected yogic asanas, pranayama, and meditation for 6 days in a week. Yoga training was imparted by a qualified yoga instructor. The training consisted of 1 practical session (60 minutes). The yogic schedule is depicted in Table 1 and Figure 2.
Anthropometric Measurements Anthropometric measurements of the subjects were taken with minimal clothing (only inner wears). Body weight in kilograms was measured using an electronic weighing machine (Delmar, India) with least count of 0.1 kg. The standing (vertical) body height in
centimeters was measured by means of an anthropometer from the sole of the feet to the vertex while standing erect. Body Mass Index (BMI) was calculated as the ratio of weight in kilograms to a height in meters squared (kg/m2).
Resting Physiological Parameters VO2 and REE were measured by portable computerized equipment (K4b2 COSMED, Italy). The standard gas mixture was used to calibrate the oxygen and carbon dioxide. Respiratory quotient (RQ) and resting CO2 elimination were also obtained from the K4b2 system. Peripheral oxygen saturation (SpO2) was measured by using pulse oximetry with a probe attached to the index finger.
Biochemical Estimation Collection of Samples. Fasting venous blood samples were collected in ethylenediamine-tetraacetic-acid-treated vials from an antecubital vein in the morning when the volunteers were in resting condition in the lying position. Just after collection, 1 portion of ethylenediaminetetraacetic acid–treated whole blood was kept in a separate vial. The aliquot of 0.5 ml of whole blood was treated with equal volume of 10% metaphosphoric acid and 1 ml 10% trichloroacetic acid (TCA) was mixed properly and kept in a separate vial to analyze vitamin C, reduced glutathione (GSH), oxidized glutathione (GSSG), and malondialdehyde (MDA). The remaining blood sample was centrifuged at 1000 g for 15 minutes to collect plasma and red blood cell (RBC) pellets. RBCs were washed twice with a cold 150 mM potassium chloride solution at 4°C. All the chemicals for biochemical estimation have been purchased from Sigma Aldrich Co. GSH and GSSG. Supernatant obtained from metaphosphoric acid
and TCA-treated whole blood was used to estimate the GSH and GSSG by using a fluorimetric method.30 A total of 10 µl supernatant
Figure 1 — Schematic representation of study design. JPAH Vol. 12, No. 4, 2015
Yoga on Metabolism and Antioxidant–Redox Status 581
Table 1 Details of Yogic Package Was Practiced by the Volunteers Cleansing processes: session = 2 min 1. Kapalbhati (Rapid shallow breathing): It is a vigorous hyperventilation practice, which involves forceful exhalation and passive inhalation.
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Yogasanas (yogic postures): session = 40 min 1. Suryanamaskar: Sun salutation in 12 different postures, 1 round 2. Padmasana: In Sanskrit, “Padma” means “lotus.” This asana in its final position resembles the petals of a lotus. 3. Yogamudra 4. Matsayasana: In Sanskrit, “matsya” mean “fish.” That is why it is known as Matsyasana (fish posture) 5. Suptapavanmuktasana: The Sanskrit word “pawan” means “wind” and the word “mukta” means “release” or “free.” Hence, this is a wind-releasing pose because it is very useful in removing wind or flatulence from the intestines and stomach. 6. Pavanmuktasana: “Pawana” means “wind” and “mukta” means to “release.” When it is practiced, it helps to release excessive gases from the body. 7. Paschimottanasana: “Paschim” means “back” and “uttan” means “stretching.” 8. Vajrasana: Sitting posture 9. Suptavajrasana 10. Gomukhasana: The interlocked hand in this asana takes the shape of Gomukh, which means a cow’s face in Sanskrit. That is why it is known as Gomukhasana. 11. Sarvangasana: In Sanskrit, “Sarvang” means “all parts of the body.” Because this asana having the effect on almost all the body parts and organs, it is called Sarvangasana. 12. Halasana: “Hala” means a “plow” in Sanskrit. The pose is called Halasana because in its practice, the body takes the shape of Indian plow. 13. Karnapedasana 14. Bhujangasana: In Sanskrit, “Bhujanga” means “cobra.” The final position of this asana resembles the “hooded snake.” Hence, it is called Bhujangasana. 15. Shavasana: In Sanskrit, “shava” means a “dead body.” Relaxed supine posture. This asana resembles a dead body that is why it is known as Shavasana. Pranayama (breathing exercises): session = 10 min 1. Bhastrika: Forceful expulsion of breathing, Bhastrika pranayama is all about inhaling and exhaling completely so that your body gets maximum amount of oxygen. 2. Anulom—vilom: Anuloma viloma is also called the alternate nostril breathing. 3. Bhramri: The word “Bhramari” is derived from “Bhramara,” which means a black bee. While practicing this Pranayama, the sound produced resembles the buzzing of a black bee with closed eyes, ears, and lips. Meditation: session = 8 min 1. Omkar meditation: Om chant
was treated with 100 µl o-phthalaldehyde at pH 8 and estimated GSH using a Fluorescence spectrophotometer with an excitation and emission wavelength of 350 nm and 420 nm, respectively. GSSG was also measured with this same procedure at pH 12 after treatment with 10 µl N-ethylmaleimide to prevent interference of GSH with the measurement of GSSG. Total Antioxidant Status (TAS). It was measured in 10-µl plasma with an additional 1.0 ml of ABTS (2,2'-Azino-di-[3ethylbenzthiazoline sulphonate]) radical cation decolorizing diluted reagent, using a Randox kit (Cat No NX 2332; Randox Laboratories, UK) Vitamin C. Supernatant obtained from metaphosphoric acid and TCA-treated whole was used to estimate vitamin C.31 Briefly, the 0.5 ml supernatant was treated with a few drops (0.050 ml) of bromine water followed by a few drops (50 µl) of thiourea to remove the excess bromine, and thus, the clear solution was obtained. Then 0.4 ml 2,4-dinitrophenyl-hydrazine solution was added to all standards and also sampled with the oxidized ascorbic acid. Total vitamin C content employing coupling reaction of 2,4-dinitrophenyl hydrazine (DNPH) was measured at 540 nm using a spectrophotometer.
Vitamin E. The estimation of vitamin E from 100 µl of plasma
using the bathophenanthroline method.32 The lipid residue obtained using 100 µl of redistilled ethanol and 500 µl of petroleum ether was redissolved in 400 µl of absolute ethanol. To this solution, 50 µl ferric chloride, 50 µl orthophosphoric acid, and 50 µl bathophenanthroline reagents were added. Vitamin E presents in the lipid residue, reduces ferric ions to ferrous ions, and forms a pink-colored complex with bathophenanthroline orthophosphoric acid. Absorption due to the pink complex was measured at 536 nm using a spectrophotometer.
Hydroperoxide Estimation (FOX 1 Assay). Hydroperoxide was estimated from plasma.33 Xylenol orange, in an acidic solution with sorbitol, and ammonium iron sulfate reacted to produce a purple color proportional to the concentration of hydrogen peroxide (H2O2) in the sample. We took 50µl of the sample plasma and added 950 µl of FOX reagent and incubate for 30 min at room temperature. We read the absorbance at a web length of 560 nm. Protein Carbonyl (PC). PC reacts with DNPH to form a Schiff
base to produce the corresponding hydrazine, which can be analyzed spectrophotometrically.34 A plasma sample (50µl) was added to 0.950 ml of DNPH and mixed thoroughly. After proper mixing,
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Figure 2 — Various yoga postures as practiced by the volunteers during yoga session.
1.0 ml of TCA was added. After 3 times washing of the precipitate obtained from above mixture, 2 ml of guanidine hydrochloride was added and optical density (OD) was measured at 366 nm web length using a spectrophotometer. Malondialdehyde (MDA). MDA content of the sample was estimated by using plasma.35 In brief, metaphosphoric acid and TCA-treated whole blood supernatant were mixed with equal volumes of thiobarbituric acid solution and incubated at 90°C for 30 minutes; color density was measured spectrophotometrically at 535 nm. Glutathione Reductase (GR). RBC lysate was used to measure
the GR activity.36 The final reaction mixture contains GSSG and NADPH; these are incubated at pH 7.5, and the change in OD was measured at 340 nm for 3 minutes at an interval of 30 seconds.
Glutathione Peroxidase (GPx). GPx was measured in RBC
pellets by using the Cayman Kit (Cat No. 703102, Cayman Chemical Laboratories, USA). Assay buffer (100 µl) and cosubstrate was added to all the wells of enzyme-linked immunosorbent assay plates. A total of 20 µl of diluted GPx control was added to the positive control wells, and 20 µl of the sample was added to the sample wells. An enzymatic reaction was initiated by adding 20 µl of cumene hydroxide. Change in OD was recorded at 60-second intervals for 300 seconds at 340 nm using an enzyme-linked immunosorbent assay reader. Calculation was made according the kit protocol.
Superoxide Dismutase (SOD). SOD was measured in RBC
pellets by using the Cayman Kit (Cat No. 706002, Cayman Chemical Laboratories). A total of 200 µl of diluted radical was added to all
wells of the plate. Ten µl of standard and 10 µl of sample added, respectively, to the standard wells and sample wells. An enzymatic reaction was initiated by adding 20 µl of xanthine oxidase. OD was measured after a 20-minute incubation at 460 nm. Calculation was made according to kit protocol. Catalase. Catalase was measured spectrophotometrically using
RBC lysate.37 Briefly, a 50 µl lysate of RBC was treated with 1.0 ml of 30-mM H2O2, and absorbance was measured at a 240-nm wavelength for 1 minute at an interval of 30 seconds.
G l u t a t h i o n e S Tr a n s fe r a s e ( G S T ) . T h e e s t i m a t i o n
of GST using 1-chloro-2,4-dinitrobenzene was carried out spectrophotometrically.38 The reaction mixture contains 0.1 ml of GSH, 20 µl of 1-chloro-2, 4-dinitrobenzene and 1 ml of a phosphate buffer. The reaction was initiated by adding 0.1 ml of RBC lysate. The change in OD at an interval of 15 seconds for 300 seconds was recorded using a spectrophotometer at 340 nm. Glucose-6-Phosphate Dehydogenase (G6PDH). The estimation
of G6PDH was carried out using RBC lysate.39 A total of 100 µl of RBC lysate was incubated with 100 mM of magnesium chloride (200 µl), glucose-6-phosphates (100 µl), nicotinamide adenine dinucleotide phosphate (100 µl), and a buffer at pH 7.4. The change in OD was measured at 340 nm for 3 minutes at an interval of 30 seconds using a spectrophotometer.
Blood Glucose. Blood glucose was estimated by using GlucoDr
(All Medicus Co., Ltd., Korea), blood glucose test meter in fasting condition between 6:30 AM and 7:15 AM, and the middle finger tip was used to take the blood.
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Yoga on Metabolism and Antioxidant–Redox Status 583
Statistical Analysis One-way analysis of variance (ANOVA) without replication to analyze significance of between-group variance and within-group therein was performed. Once Schiff’s F showed P ≤ .05, data underwent the Tukey–Kramer post hoc test for within-group significance, using GraphPad InStat Version 3.00 for Windows (GraphPad Software, San Diego, CA, USA). Data was expressed as mean ± standard error of the mean (SEM). P ≤ .05 was considered to be statically significant.
Results
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Anthropometric Data Anthropometric data are summarized in Table 2. Body weight (kg) and BMI (kg/m2) did not show any significant change before and after yogic practices, though a nonsignificant reduction in both these parameters was observed in the yoga group, whereas there was an increase in these parameters the control group following the period of yogic practice. No significant difference was found between the baseline data of the control group and yoga group.
Resting Physiological Variables Resting physiological variables are illustrated in Table 3. VO2 and REE were decreased in the yoga group following 3 months of yogic practices. On the other hand, RQ (P < .01) and resting CO2 elimination (P < .001) decreased significantly as a result of yogic practices. SpO2 was also significantly increased following 3 months of yogic practice. No significant change was noted in the control group following yogic practice. No significant difference was noted between baseline data of the control group and yoga group in these resting physiological parameters.
Enzymatic and Nonenzymatic Antioxidant and Blood Glucose Nonenzymatic antioxidant variables and blood sugar are depicted in Table 4. The levels of GSH were increased significantly (P < .001), with a decrease in GSSG (P < .01). This led to a significant (P < .001) increase in the GSH and GSSG ratio following 3 months of yogic practices. TAS, an important antioxidant marker, increased significantly (P < .001) following 3 months of yogic practice. The levels of vitamin C and vitamin E were also increased significantly (P < .001) following yogic practices. A nonsignificant decrease in MDA levels of the yoga group was noted. No significant changes were observed in levels of hydroperoxides measured using FOX , PC, and blood glucose levels following yogic practice, although a nonsignificant reduction in these parameters was observed. The control group did not show any significant change in nonenzymatic antioxidant variables following yogic practice. No significant difference was found between baseline data of the control group and yoga group in enzymatic and nonenzymatic antioxidant parameters and levels of blood glucose. Activities of antioxidant enzymes are summarized in Figure 3. SOD and GST activity increased significantly (P < .001), while GPx activity decreased significantly (P < .001). GR activity also increased significantly (P < .05) following yogic practice. No significant change was noted in these parameters in the control group. The catalase and G6PDH activity in the yoga group increased nonsignificantly as a result of yogic practice.
Discussion Body weight and BMI did not show any significant change in the current study, though there was a nonsignificant reduction in the mean values of both parameters in the yoga group. There are
Table 2 Physical Characteristics of Subjects Control group (n = 30) Age (years) Height (cm) Body weight (kg) Body mass index (kg/m2)
Yoga group (n = 34)
Before
After
Before
After
37.5 ± 1.39 171.7 ± 2.95 81.1 ± 6.79 27.4 ± 1.43
— — 81.4 ± 7.32 27.5 ± 1.68
40.9 ± 0.90 169.5 ± 0.90 73.8 ± 8.02 25.7 ± 0.43
— — 73.1 ± 8.20 25.4 ± 0.44
Note. Values are expressed as mean ± SEM.
Table 3 Physiological Changes of Control and Yoga Group Before and After 3 Months Control group (n = 30) Resting O2 consumption (ml/min) Resting CO2 elimination (ml/min) Respiratory quotient Resting energy expenditure (kj/min) Peripheral oxygen saturation (%)
Yoga group (n = 34)
Before
After
Before
After
238.5 ± 13.7 220.8 ± 11.6 0.92 ± 0.04 4.79 ± 0.24 97.3 ± 0.26
276.9 ± 17.68 229.6 ± 18.95 0.88 ± 0.03 5.53 ± 0.20 97.3 ± 0.42
240.7 ± 12.9 227.5 ± 13.1 0.95 ± 0.02 4.97 ± 0.27 96.3 ± 0.41
210.4 ± 8.8 173. 1 ± 8.2** 0.82 ± 0.02*** 4.20 ± 0.18 97.6 ± 0.15**
Note. Values are expressed as mean ± SEM. ** P < .01, as compared with yoga group base line data; *** P < .001, as compared with yoga group base line data. No significant difference was noted between base line data of control group and yoga group. JPAH Vol. 12, No. 4, 2015
Table 4 Antioxidant, Oxidative Stress Markers, and Blood Glucose of Control and Yoga Group Before and After 3 Months Control group (n = 30) GSH (nmol/ml) GSSG (nmol/ml) GSH/GSSG TAS (mmol/ml) Vitamin C (mg/dl)
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Vitamin E (μg/ml) Hydroperoxides (μmol/ml) MDA (nmol/ml) PC (nmol/ml) Blood glucose (mg/dl)
Yoga group (n = 34)
Before
After
Before
After
260.2 ± 17.3 399.1 ± 10.13 0.65 ± 0.06 2.66 ± 0.15 1.37 ± 0.08 12.24 ± 1.97 506.1 ± 37.1 8.56 ± 0.63 118.6 ± 12.71 101.3 ± 5.3
264.7 ± 15.6 392.7 ± 14.1 0.67 ± 0.07 2.78 ± 0.09 1.34 ± 0.06 11.80 ± 2.73 509.4 ± 36.9 8.38 ± 0.60 117.6 ± 13.3 101.7 ± 3.9
292.4 ± 18.5 394.9 ± 19.4 0.98 ± 0.25 2.69 ± 0.06 1.44 ± 0.08 13.02 ± 0.90 545.1 ± 50.6 7.78 ± 0.50 114.2 ± 17.8 96.4 ± 1.6
457.5 ± 16.7 *** 300.6 ± 18.3** 1.86 ± 0.21*** 3.19 ± 0.04*** 3.35 ± 0.12*** 19.62 ± 0.38*** 452.6 ± 60.2 6.01 ± 0.46 106.4 ± 9.7 95.7 ± 9.7
Note. Values are expressed as mean ± SEM. ** P < .01, as compared with yoga group base line data; *** P < .001, as compared with yoga group base line data. Abbreviations: GSH, reduced glutathione; GSSG, oxidized glutathione; GSH/GSSG, ratio of reduced and oxidized glutathione; TAS, total antioxidant status; MDA, malondialdehyde; PC, protein carbonyl.
Figure 3 — Changes in enzymatic activity of control and yoga group before and after 3 months. (A) Super oxide dismutase, (B) catalase, (C) glutathione peroxidase, (D) glutathione reductase, (E) glutathione 6 phosphate dehydrogenase, and (F) glutathione S-transferase. Values are expressed as mean ± SEM. ** P < .01, as compared with yoga group base line data; *** P < .001, as compared with yoga group base line data.
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Yoga on Metabolism and Antioxidant–Redox Status 585
conflicting reports in body weight changes after yogic exercises in the literature, as both a decrease and an increase are reported.8,40 A possible explanation could be that the participants of the current study were trained Air Force personnel and were habituated to perform routine physical exercises. During the study, the yoga group performed yoga training in place of exercise. The VO2, resting CO2 elimination, RQ, and REE decreased as a result of yogic practice. Various studies showed that relaxation techniques decreased O2 consumption, CO2 elimination, and respiration rate.41,42 Yoga may have a beneficial role to calm down the body and mind, which decreases O2 consumption as well as CO2 elimination along with decreasing REE as a relaxation response. The SpO2 of the body increased due to yogic practices, and there was no such change observed in the control group. This may due to the breathing maneuvers such as Kapalbhati, Bhastrika, Anulom-vilom, and Bhramari.43,44 This study shows that SOD activity increases significantly, and there is a trend to increase in catalase activity in the yoga group, whereas SOD activity decreases in the control group. Earlier studies have reported that the yogic practice helped in the increase of SOD activity.21 Yogic practices decreased the activity of GPx and increased activity of GR. The GSH level was increased significantly in the yoga group; the ratio of reduced to oxidized glutathione (GSH/ GSSG ratio) is another important and sensitive marker of the antioxidant system that has been shown to increase significantly in the yoga group. These may be due to improvement of the antioxidant defense mechanism by yoga.18 H2O2 is generated directly from superoxide (O2–) though a rapid dismutation reaction that can occur either enzymatically with SOD or spontaneously. This means that wherever O2– is generated, there is also the formation of H2O2. SOD and catalase are metalloproteins that catalyze O2– and H2O2, respectively. SOD catalyzes the formation of H2O2 from 2 O2–, whereas catalase catalyzes the formation of oxygen and water from an H2O2 molecule. Increments of SOD activity produced more H2O2. Catalase activity was increased, perhaps because yogic practice catalyzed more H2O2 and produced O2 and water. Hence, hydroperoxide levels were decreased in the yoga group. GPx catalyzes the reduction of hydroperoxides, including H2O2, and functions to protect the cell from oxidative damage. GSSG, produced upon reduction of hydroperoxide by GPx, is recycled to its reduced state by GR using NADPH as a hydrogen donor. Here, the production of NADPH is coupled with G6PDH. This study showed that yoga increased GSH and decreased GSSG by altering the activity of GPx, GR, and G6PDH. An earlier report also supports this observation.18 GSTs are multifunctional enzymes, which play a key role in cellular detoxification. The enzymes protect cells against toxicants by conjugating them with glutathione, thereby neutralizing their electrophilic sites and rendering the products more water-soluble.45 The glutathione conjugates are metabolized further to mercapturic acid and then excreted. In this study, GST activity increased significantly after 3 months of yogic practices. This also acts as a defense mechanism of the body against reactive oxygen species production. GST also acts to catalyze the reaction between the thiol group of GSH and possible alkylating agents, allowing GSH to carry out its detoxifying function. Levels of vitamins C and E increased in the yoga group. Vitamin C and vitamin E have a profound effect on the body; during lipid peroxidation, vitamins function as a potent chain-breaking antioxi-
dant, intercepting lipid peroxyl radicals and forming the vitamin E radical as a product. Vitamin C acts to generate vitamin E by accepting the electron from the vitamin E radical, with the vitamin C radical being formed, and either in the urine or regenerated to vitamin C via electron donation from GSH. It also reported that both vitamin E and C function to scavenge superoxide radical and hydroxyl radical in lipid and aqueous phase, respectively, inhibiting lipid peroxidation and oxidative damage to other macromolecules. Without changing any diet pattern, vitamin C and vitamin E were increasing significantly in the yoga group. This may be due to an increase in total antioxidant status or shifting of the volunteers toward reducing redox status. TAS has been seen to improve significantly after yogic practice, denoting a marked improvement in the overall cellular antioxidant level. Studies reported that the yogic practice helps in the improvement of TAS.18 Another study also showed that diaphragmatic breathing/breathing maneuvers also increased biological antioxidant potential and reduced exercise-induced oxidative stress.17 Oxidative stress generally causes damage to the membrane polyunsaturated fatty acids leading to the generation of MDA, a thiobarbituric-acid-reacting substance. This biochemical study implies that the products of lipid peroxidation decrease with yogic practice. This is also supported by several previous studies.20–22 Studies also showed that the yogic practice decreased MDA level in diabetic patients.21 Sudarshan Kriya yoga has been reported to be beneficial in decreasing MDA level.46 Protein carbonyl also decreased due to yogic practice indicating prevention of protein oxidation. Blood sugar levels decreased in the yoga group after 3 months of yogic practice, whereas no changes were observed in the blood sugar levels of the control group. There are reports suggesting that blood sugar levels decreased in the diabetic patient with yogic practices.22
Conclusion This study showed that yoga has the beneficial role to decrease oxidative stress by improving enzymatic defense mechanism and nonenzymatic antioxidants. It also maintains antioxidant and redox status. Yoga has also a beneficial role in relaxing the body by decreasing VO2 and REE. Acknowledgments Sincere gratitude to the director of the Defence Institute of Physiology and Allied Sciences (DIPAS) in Delhi, India, for giving necessary permission to conduct this study. We are thankful to Director Morarji Desai, National Institute of Yoga (MDNIY), New Delhi. We are also grateful to volunteers of this study for their kind help in the successful completion of the study. We acknowledge the support of staff members of the CARTY and nutrition division of DIPAS. The study was financially supported by DIPAS and MDNIY.
References 1. Pal R, Saha M. Role of yogic practice on physical health: a review. Indian J Appl Research. 2013;3(4):34–36. 2. Field T. Yoga clinical review. Complement Ther Clin Pract. 2011;17(1):1–8. PubMed 3. Sengupta P. Health impacts of yoga and pranayama: a state-of-the-art review. Int J Prev Med. 2012;3(7):444–458. PubMed
JPAH Vol. 12, No. 4, 2015
Downloaded by Washington Univ In St Louis on 09/16/16, Volume 12, Article Number 4
586 Pal et al 4. Chan W, Immink MA, Hillier S. Yoga and exercise for symptoms of depression and anxiety in people with poststroke disability: a randomized, controlled pilot trial. Altern Ther Health Med. 2012;18(3):34–43. PubMed 5. Chung SC, Brooks MM, Rai M, Balk JL, Rai S. Effect of sahaja yoga meditation on quality of life, anxiety, and blood pressure control. J Altern Complement Med. 2012;18(6):589–596. PubMed doi:10.1089/ acm.2011.0038 6. Gupta SS, Sawane MV. A comparative study of the effects of yoga and swimming on pulmonary functions in sedentary subjects. Int J Yoga. 2012;5(2):128–133. PubMed doi:10.4103/0973-6131.98232 7. Balasubramanian B, Pansare MS. Effect of yoga on aerobic and anaerobic power of muscles. Indian J Physiol Pharmacol. 1991;35(4):281– 282. PubMed 8. Bera TK, Rajapurkar MV. Body composition, cardiovascular endurance and anaerobic power of yogic practitioner. Indian J Physiol Pharmacol. 1993;37(3):225–228. PubMed 9. Ray US, Sinha B, Tomer OS, Pathak A, Dasgupta T, Selvamurthy W. Aerobic capacity & perceived exertion after practice of Hatha yogic exercises. Indian J Med Res. 2001;114:215–221. PubMed 10. Ray US, Mukhopadhyaya S, Purkayastha SS, et al. Effect of yogic exercises on physical and mental health of young fellowship course trainees. Indian J Physiol Pharmacol. 2001;45(1):37–53. PubMed 11. Streeter CC, Gerbarg PL, Saper RB, Ciraulo DA, Brown RP. Effects of yoga on the autonomic nervous system, gamma-aminobutyric-acid, and allostasis in epilepsy, depression, and post-traumatic stress disorder. Med Hypotheses. 2012;78(5):571–579. PubMed doi:10.1016/j. mehy.2012.01.021 12. Muralikrishnan K, Balakrishnan B, Balasubramanian K, Visnegarawla F. Measurement of the effect of Isha Yoga on cardiac autonomic nervous system using short-term heart rate variability. J Ayurveda Integr Med. 2012;3(2):91–96. PubMed doi:10.4103/0975-9476.96528 13. Selvamurthy W, Sridharan K, Ray US, et al. A new physiological approach to control essential hypertension. Indian J Physiol Pharmacol. 1998;42(2):205–213. PubMed 14. Murugesan R, Govindarajulu N, Bera TK. Effect of selected yogic practices on the management of hypertension. Indian J Physiol Pharmacol. 2000;44(2):207–210. PubMed 15. Yadav RK, Magan D, Mehta N, Sharma R, Mahapatra SC. Efficacy of a short-term yoga-based lifestyle intervention in reducing stress and inflammation: preliminary results. J Altern Complement Med. 2012;18(7):662–667. PubMed doi:10.1089/acm.2011.0265 16. Kiecolt-Glaser JK, Christian L, Preston H, et al. Stress, inflammation, and yoga practice. Psychosom Med. 2010;72(2):113–121. PubMed doi:10.1097/PSY.0b013e3181cb9377 17. Martarelli D, Cocchin M, Scuri S, Pompei P. Diaphragmatic breathing reduces exercise-induced oxidative stress. Evid Based Complement Alternat Med. 2011;932430. doi:10.1093/ecam/nep169 18. Sinha S, Singh SN, Monga YP. Ray US. Improvement of glutathione and total antioxidant status with yoga. The J Alt Com Med. 2007;13(10):1085–1090. doi:10.1089/acm.2007.0567 19. Yadav RK, Ray RB, Vempati R, Bijlani R. Effect of a comprehensive yoga based lifestyle modification programme on lipid peroxidation. Indian J Physiol Pharmacol. 2005;49(3):358–362. PubMed 20. Bhattacharya S, Pandey US, Verma NS. Improvement in oxidative status with yogic breathing in young healthy males. Indian J Physiol Pharmacol. 2002;46:349–354. PubMed 21. Gordon LA, Morrison EY, McGrowder DA, et al. Effect of exercise therapy on lipid profile and oxidative stress indicators in patients with type 2 diabetes. BMC Complement Altern Med. 2008;8:21. PubMed doi:10.1186/1472-6882-8-21
22. Malhotra V, Singh S, Sharma SB, et al. The status of NIDDM patients after yoga asanas: assessment of important parameters. J Clin and Diagno Res. 2010;4:2652–2667. 23. Singh S, Kyizom T, Singh KP, Tandon OP, Madhu SV. Influence of pranayamas and yoga: asanas on serum insulin, blood glucose and lipid profile in type 2 diabetes. Indian J Clin Biochem. 2008;23(4):365–368. PubMed doi:10.1007/s12291-008-0080-9 24. Venditti P, Masullo P, Di Meo S. Effect of training on H2O2 release by mitochondria from rat skeletal muscle. Arch Biochem Biophys. 1999;372(2):315–320. PubMed doi:10.1006/abbi.1999.1494 25. Leeuwenburgh C, Fiebig R, Chandwaney R, Ji LL. Aging and exercise training in skeletal muscle: responses of glutathione and antioxidant enzyme systems. Am J Physiol. 1994;267(2 Pt 2):439–445. PubMed 26. Leeuwenburgh C, Hollander J, Leichtweis S, Griffiths M, Gore M, Ji LL. Adaptations of glutathione antioxidant system to endurance training are tissue and muscle fiber specific. Am J Physiol. 1997;272(1 Pt 2):363–369. PubMed 27. Powers SK, Criswell D, Lawler J, et al. Influence of exercise and fiber type on antioxidant enzyme activity in rat skeletal muscle. Am J Physiol. 1994;266(2 Pt 2):375–380. PubMed 28. Powers SK, Criswell D, Lawler J, et al. Rigorous exercise training increases superoxide dismutase activity in ventricular myocardium. Am J Physiol. 1993;265(6 Pt 2):2094–2098. PubMed 29. Somani SM, Frank S, Rybak LP. Responses of antioxidant system to acute and trained exercise in rat heart subcellular fractions. Pharmacol Biochem Behav. 1995;51(4):627–634. PubMed doi:10.1016/0091-3057(94)00427-K 30. Hissin PJ, Hiff R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 1976;74(1):214– 226. PubMed doi:10.1016/0003-2697(76)90326-2 31. Khan MMR, Rahman MM, Islam MS, Begum SA. A simple ultraviolet—spectrometric method for the determination of vitamin C content in various fruits and vegetables at sylhet area in Bangladesh. J Biol Sci. 2006;6(2):388–392. doi:10.3923/jbs.2006.388.392 32. Desai ID. Vitamin E analysis method for animal tissues. Methods Enzymol. 1984;105:138–147. PubMed doi:10.1016/S00766879(84)05019-9 33. Wolff SP. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol. 1994;233:182–189. doi:10.1016/S0076-6879(94)33021-2 34. Reznick AZ, Packer L. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Methods Enzymol. 1994;233:357– 363. PubMed doi:10.1016/S0076-6879(94)33041-7 35. Ohkawa H. Assay of lipid peroxide in animal tissue by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351–358. PubMed doi:10.1016/0003-2697(79)90738-3 36. Racker E. Glutathione reductase from baker’s yeast and beef liver. J Biol Chem. 1955;217(2):855–865. PubMed 37. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–126. PubMed doi:10.1016/S0076-6879(84)05016-3 38. Habig WH, Jakoby WB. Assay for differentiation of glutathione S-transferase. Methods Enzymol. 1981;77:398–405. PubMed doi:10.1016/ S0076-6879(81)77053-8 39. Bernardi L, Passino C, Spadacini G, et al. Reduced hypoxic ventilatory response with preserved blood oxygenation in yoga trainees and Himalayan Buddhist monks at altitude: evidence of a different adaptive strategy? Eur J Appl Physiol. 2007;99(5):511–518. PubMed doi:10.1007/s00421-006-0373-8 40. Kesterson J, Clinch NF. Metabolic rate, respiratory exchange ratio, and apneas during meditation. Am J Physiol. 1989;256(3 Pt 2):632–638. PubMed
JPAH Vol. 12, No. 4, 2015
Yoga on Metabolism and Antioxidant–Redox Status 587 44. Mason H, Vandoni M, Barbieri G, Codrons E, Ugargol V, Bernardi L. Cardiovascular and respiratory effect of yogic slow breathing in the yoga beginner: what is the best approach? Evid Based Complement Alternat Med. 2013; 743504. doi:10.1155/2013/743504 45. Habig WH, Pabst MJ, Jakoby WB, Glutathione S. Transferases the first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249(22):7130–7139. PubMed 46. Agte VV, Tarwadi K. Sudarshan kriya yoga for treating type 2 diabetes: a preliminary study. Altern Complement Ther. 2004;10(4):220–222. doi:10.1089/1076280041580323
Downloaded by Washington Univ In St Louis on 09/16/16, Volume 12, Article Number 4
41. Warrenburg S, Pagano RR, Woods M, Hlastala M. A comparison of somatic relaxation and EEG activity in classical progressive relaxation and transcendental meditation. J Behav Med. 1980;3(1):73–93. PubMed doi:10.1007/BF00844915 42. Boyland E, Chasseaud LF. The role of glutathione and glutathione S-transferases in mercapturic acid biosynthesis. Adv Enzymol. 1969;32:173–219. PubMed 43. Ashok C. Impact of asanas and pranayama on blood oxygen saturation level. Br J Sports Med. 2010;44:i69. doi:10.1136/ bjsm.2010.078725.228
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